Exon duplications in the ATP7Agene: Frequency and Transcriptional Behaviour

Mogensen Mie, Skjørringe Tina, Kodama Hiroko, Silver Kenneth, Horn Nina, Møller

Description

Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene. Methods The ATP7A gene was screened for exon duplications using multiplex ligation-dependent probe amplification (MLPA). The expression level of ATP7A was investigated by real-time PCR and detailed analysis of the ATP7A mRNA was performed by RT-PCR followed by sequencing. In order to investigate whether the identified duplicated fragments originated from a single or from two different X-chromosomes, polymorphic markers located in the duplicated fragments were analyzed. Results Partial ATP7A gene duplication was identified in 20 unrelated patients including one patient with Occipital Horn Syndrome (OHS). Duplications in the ATP7A gene are estimated from our material to be the disease causing mutation in 4% of the Menkes disease patients. The duplicated regions consist of between 2 and 15 exons. In at least one of the cases, the duplication was due to an intra-chromosomal event. Characterization of the ATP7A mRNA transcripts in 11 patients revealed that the duplications were organized in tandem, in a head to tail direction. The reading frame was disrupted in all 11 cases. Small amounts of wild-type transcript were found in all patients as a result of exon-skipping events occurring in the duplicated regions. In the OHS patient with a duplication of exon 3 and 4, the duplicated out-of-frame transcript coexists with an almost equally represented wild-type transcript, presumably leading to the milder phenotype. Conclusions In general, patients with duplication of only 2 exons exhibit a milder phenotype as compared to patients with duplication of more than 2 exons. This study provides insight into exon duplications in the ATP7A gene.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 31
• Langue: English

Extrait

Mogensenet al.Orphanet Journal of Rare Diseases2011,6:73 http://www.ojrd.com/content/6/1/73

R E S E A R C H

Open Access

Exon duplications in theATP7Agene: Frequency and Transcriptional Behaviour 1†1†1 1*2 3 Mie Mogensen , Tina Skjørringe , Hiroko Kodama , Kenneth Silver , Nina Horn and Lisbeth B Møller

Abstract Background:Menkes disease (MD) is an Xlinked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in theATP7Agene. Thirtythree Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in theATP7Agene. Methods:TheATP7Agene was screened for exon duplications using multiplex ligationdependent probe amplification (MLPA). The expression level ofATP7Awas investigated by realtime PCR and detailed analysis of the ATP7AmRNA was performed by RTPCR followed by sequencing. In order to investigate whether the identified duplicated fragments originated from a single or from two different Xchromosomes, polymorphic markers located in the duplicated fragments were analyzed. Results:PartialATP7Agene duplication was identified in 20 unrelated patients including one patient with Occipital Horn Syndrome (OHS). Duplications in theATP7Agene are estimated from our material to be the disease causing mutation in 4% of the Menkes disease patients. The duplicated regions consist of between 2 and 15 exons. In at least one of the cases, the duplication was due to an intrachromosomal event. Characterization of theATP7A mRNA transcripts in 11 patients revealed that the duplications were organized in tandem, in a head to tail direction. The reading frame was disrupted in all 11 cases. Small amounts of wildtype transcript were found in all patients as a result of exonskipping events occurring in the duplicated regions. In the OHS patient with a duplication of exon 3 and 4, the duplicated outofframe transcript coexists with an almost equally represented wildtype transcript, presumably leading to the milder phenotype. Conclusions:In general, patients with duplication of only 2 exons exhibit a milder phenotype as compared to patients with duplication of more than 2 exons. This study provides insight into exon duplications in theATP7Agene.

Background Menkes disease (MD; MIM# 309400) is a multisystemic lethal disorder of impaired copper metabolism due to mutations in the XlinkedATP7Agene [1,2]. The disor der is transmitted in an Xlinked recessive pattern. The ATP7A protein is a member of the Ptype ATPase family that ensures the ATPdriven translocation of metal cations across cellular membranes. The protein plays a dual role: it is responsible for the copperloading of several copperrequiring enzymes, as well as for the ATPdriven efflux of copper from the cell [35]. At nor mal physiological copper concentrations, ATP7A is

* Correspondence: [e-mail] †Contributed equally 1 Center for Applied Human Molecular Genetics, Kennedy Center, Gl. Landevej 7, 2600 Glostrup Denmark Full list of author information is available at the end of the article

localized to the transGolgi network (TGN) [3] where copperloading of enzymes in the secretory pathway takes place. In response to the increase in copper con centration, the protein is translocated to the plasma membrane [3] where it is involved in pumping excess copper out of the cell. In the human body, copper is taken up in the gastrointestinal tract. However, patients with MD are unable to transport copper further from the intestinal cells, and less copper is therefore delivered to the blood. These patients have severe developmental and neurological impairments due to a subnormal amount of copper in the brain. In addition, a reduced activity of several copperdependent enzymes can lead to a variety of symptoms such as connective tissue abnormalities, tortuosity of blood vessels and peculiar hair (kinky, steely hair or pili torti) [1,2]. The phenotypic features of MD can be divided into at least three

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